PROJECT SUMMARY DNA double strand breaks (DSBs) are potentially catastrophic cellular insults that must be recognized and repaired to maintain genome integrity. DSB repair (DSBR) depends on two complicated and highly coordinated pathways: non-homologous end joining (NHEJ), which is error prone, and homology recombination (HR), which is error free. DNA DSB end resection initiates HR and is a critical determinant of DNA DSB repair pathway choice; however, the precise mechanisms by which DNA DSB repair is regulated are still not well understood. Due to the role of DNA DSB repair in mediating resistance to many types of cancer treatments, including cancer ionizing radiation (IR) and chemotherapy, elucidating how cells direct the repair of DNA DSBs also has significant clinical implications for developing better diagnostic and/or therapeutic tools. Here, a novel role for SAM domain and HD domain 1 (SAMHD1) in DNA DSBR is shown. Mutations in SAMHD1 are associated with AIDS, (AGS), and (CLL). SAMHD1 possesses both dNTP triphosphohydrolase (dNTPase) and exonuclease activities. Its dNTPase activity is well established and has a role in restricting HIV-1 replication by depleting nucleotides required for reverse transcription. However, how SAMHD1 functions as a nuclease is not well understood. Preliminary data show that, in response to DNA damage, SAMHD1 forms foci at DNA damage sites and that SAMHD1 interacts with known DSBR proteins. Depletion of SAMHD1 in cells impairs DNA end resection and HR. Interestingly, depletion of SAMHD1 sensitizes cells to IR and PARP inhibitor, further suggesting its role in DSBR, but mediates resistance to HU treatment, suggesting that SAMHD1 may function as a critical mediator of resistance to distinct types of DNA damaging agents. The hypothesis being tested is that SAMHD1 maintains genome integrity in response to DNA DSBs at least in part by promoting homologous recombination through DNA end resection. Furthermore, SAMHD1 through its unique combination of exonuclease and dNTPase activities may play a critical role in discriminating resistance to IR and chemotherapy, which may be exploited for cancer therapy. To test this hypothesis, following specific aims are proposed: 1) Determine the mechanism by which SAMHD1 functions in DNA DSB repair; 2) Delineate the mechanism by which SAMHD1 is regulated in DNA DSB repair; 3) Determine the extent to which SAMHD1 functions as a biomarker for discriminating DNA damage sensitivity. These aims will be completed by a combination of cell biological, genetic, and biochemical approaches. Completion of these aims will provide novel insights into how SAMHD1 maintains genome integrity by promoting HR and prevents disease, including AGS, CLL, and HIV1 infection. Moreover, given the unique ability of SAMHD1 to regulate cellular dNTP pools and metabolize DNA, SAMHD1 may also function as an important biomarker for discriminating resistance of cancer cells to different types of cancer therapies that induce DNA damage, including chemotherapy and IR. Aicardi-Goutires syndrome chronic lymphocytic leukemia